Transfer RNAs, like other cellular RNA species in both microbial and higher systems, arise via the processing of primary transcription products in a complex series of maturation events. Now recent studies in a number of eukaryotic organisms have revealed the literally amazing finding that added to this list must be internal excision and ligation of so-called 'intervening' RNA sequences. The non-colinearity of gene and RNA transcript challenges many of our most basic concepts of molecular biology. The ubiquity and functional significance of intervening sequences are for now almost totally obscure. We propose to approach these questions as they apply to tRNA biosynthesis in the yeast S. cerevisiae. Our rationale is essentially that which has been exploited so profitably in the analysis of prokaryotic tRNA processing: the use of nonsense suppressors to determine the requirements for functional expression of a tRNA species by the isolation and characterization of mutants. Our previous experience with T4 tRNA biosynthesis has convinced us that proper recognition of tRNA precursors by the processing apparatus is a crucial step in the ultimate expression of the tRNA gene. We predict a similar conclusion for eukaryotic tRNA biosynthesis. While employing genetics as a probe to dissect the general requirements of tRNA processing, we are particularly interested in the analysis of nonsense suppressor tRNAs synthesized via precursors containing intervening sequences, In this way we hope to elucidate the presumably profound consequences of RNA processing for the regulation of gene expression in eukaryotes.